The present invention generally relates to a measuring cell for an ion cyclotron resonance mass spectrometer (FTMS) and to methods for the analysis of samples by mass spectrometry. The apparatus and methods for ion transport and analysis described herein are enhancements of the techniques referred to in the literature relating to mass spectrometry—an important tool in the analysis of a wide range of chemical compounds. Specifically, mass spectrometers can be used to determine the molecular weight of sample compounds. The analysis of samples by mass spectrometry consists of three main steps—formation of gas phase ions from sample material, mass analysis of the ions to separate the ions from one another according to ion mass, and detection of the ions. A variety of means and methods exist in the field of mass spectrometry to perform each of these three functions. The particular combination of the means and methods used in a given mass spectrometer determine the characteristics of that instrument.
To mass analyze ions, for example, one might use magnetic (B) or electrostatic (E) analysis, wherein ions passing through a magnetic or electrostatic field will follow a curved path. In a magnetic field, the curvature of the path will be indicative of the momentum-to-charge ratio of the ion. In an electrostatic field, the curvature of the path will be indicative of the energy-to-charge ratio of the ion. If magnetic and electrostatic analyzers are used consecutively, then both the momentum-to-charge and energy-to-charge ratios of the ions will be known and the mass of the ion will thereby be determined. Other mass analyzers are the quadrupole (Q), the ion cyclotron resonance (ICR), the time-of-flight (TOF), the Orbitrap™, and the quadrupole ion trap analyzers. The analyzer used in conjunction with the method described here may be any of a variety of these.
Before mass analysis can begin, gas phase ions must be formed from a sample material. If the sample material is sufficiently volatile, ions may be formed by electron ionization (EI) or chemical ionization (CI) of the gas phase sample molecules. Alternatively, for solid samples (e.g., semiconductors, or crystallized materials), ions can be formed by desorption and ionization of sample molecules by bombardment with high energy particles. Further, Secondary Ion Mass Spectrometry (SIMS), for example, uses keV ions to desorb and ionize sample material. In the SIMS process a large amount of energy is deposited in the analyte molecules, resulting in the fragmentation of fragile molecules. This fragmentation is undesirable in that information regarding the original composition of the sample (e.g., the molecular weight of sample molecules) will be lost.
For more labile, fragile molecules, other ionization methods now exist. The plasma desorption (PD) technique was introduced by Macfarlane et al. (R. D. Macfarlane, R. P. Skowronski, D. F. Torgerson, Biochem. Biophys. Res Commoun. 60 (1974) 616)(“McFarlane”). Macfarlane discovered that the impact of high energy (MeV) ions on a surface, like SIMS would cause desorption and ionization of small analyte molecules. However, unlike SIMS, the PD process also results in the desorption of larger, more labile species (e.g., insulin and other protein molecules).
Additionally, lasers have been used in a similar manner to induce desorption of biological or other labile molecules. See, for example, Cotter et al. (R. B. VanBreeman, M. Snow, R. J. Cotter, Int. J. Mass Spectrom. Ion Phys. 49 (1983) 35; Tabet, J. C.; Cotter, R. J., Tabet, J. C., Anal. Chem. 56 (1984) 1662; or R. J. Cotter, P. Demirev, I. Lys, J. K. Olthoff, J. K.; Lys, I.: Demirev, P.: Cotter et al., R. J., Anal. Instrument. 16 (1987) 93). Cotter modified a CVC 2000 time-of-flight mass spectrometer for infrared laser desorption of non-volatile biomolecules, using a Tachisto (Needham, Mass.) model 215G pulsed carbon dioxide laser. The plasma or laser desorption and ionization of labile molecules relies on the deposition of little or no energy in the analyte molecules of interest.
The use of lasers to desorb and ionize labile molecules intact was enhanced by the introduction of matrix assisted laser desorption ionization (MALDI) (K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshica, Rapid Commun. Mass Spectrom. 2 (1988) 151 and M. Karas, F. Hillenkamp, Anal. Chem. 60 (1988) 2299). In the MALDI process, an analyte is dissolved in a solid, organic matrix. Laser light of a wavelength that is absorbed by the solid matrix but not by the analyte is used to excite the sample. Thus, the matrix is excited directly by the laser, and the excited matrix sublimes into the gas phase carrying with it the analyte molecules. The analyte molecules are then ionized by proton, electron, or cation transfer from the matrix molecules to the analyte molecules. This process (i.e., MALDI) is typically used in conjunction with time-of-flight mass spectrometry (TOFMS) and can be used to measure the molecular weights of proteins in excess of 100,000 Daltons.
Further, Atmospheric Pressure Ionization (API) includes a number of ion production means and methods. Typically, analyte ions are produced from liquid solution at atmospheric pressure. One of the more widely used methods, known as electrospray ionization (ESI), was first suggested by Dole et al. (M. Dole, L. L. Mack, R. L. Hines, R. C. Mobley, L. D. Ferguson, M. B. Alice, J. Chem. Phys. 49, 2240, 1968). In the electrospray technique, analyte is dissolved in a liquid solution and sprayed from a needle. The spray is induced by the application of a potential difference between the needle and a counter electrode. The spray results in the formation of fine, charged droplets of solution containing analyte molecules. In the gas phase, the solvent evaporates leaving behind charged, gas phase, analyte ions. This method allows for very large ions to be formed. Ions as large as 1 MDa have been detected by ESI in conjunction with mass spectrometry (ESMS).
In addition to ESI, many other ion production methods might be used at atmospheric or elevated pressure. For example, MALDI has recently been adapted by Laiko et al. to work at atmospheric pressure (Victor Laiko and Alma Burlingame, “Atmospheric Pressure Matrix Assisted Laser Desorption”, U.S. Pat. No. 5,965,884, and Atmospheric Pressure Matrix Assisted Laser Desorption Ionization, poster #1121, 4th International Symposium on Mass Spectrometry in the Health and Life Sciences, San Francisco, Aug. 25-29, 1998) and by Standing et al. at elevated pressures (Time of Flight Mass Spectrometry of Biomolecules with Orthogonal Injection+Collisional Cooling, poster #1272, 4th International Symposium on Mass Spectrometry in the Health and Life Sciences, San Francisco, Aug. 25-29, 1998; and Orthogonal Injection TOFMS Anal. Chem. 71(13), 452A (1999)). The benefit of adapting ion sources in this manner is that the ion optics (i.e., the electrode structure and operation) in the mass analyzer and mass spectral results obtained are largely independent of the ion production method used.
Many different types of analyzers have been used to mass analyze sample ions. One important type of mass analyzer is the Fourier transform ion cyclotron (FTICR) mass analyzer. In FTICR-MS the mass-to-charge ratios m/z of ions are measured by their cyclotron movements in a homogeneous magnetic field with high field strength. The magnetic field is usually generated by superconductive magnetic coils cooled in liquid helium. Nowadays, such magnet coils provide usable cell diameters of around 6 to 12 centimeters at magnetic field strengths of 7 to 12 Tesla.
The orbital frequency of the ions (ion cyclotron frequency) is measured in ICR measuring cells located within the homogeneous part of the magnetic field. The ICR measuring cells normally comprise four longitudinal electrodes which extend in a cylindrical arrangement parallel to the magnetic field lines and surround the measuring cell like a sliced sleeve. Usually, two of these electrodes are used to bring ions, introduced close to the axis, into their cyclotron orbits (into their cyclotron motion), ions with the same mass-to-charge ratio being excited as in phase as possible in order to obtain a synchronously orbiting cloud of ions. The two other electrodes serve to measure the orbiting of the ion clouds by their image currents, which are induced in the electrodes as the ions fly past. The term “image currents” is normally used even though it is actually the induced “image voltages” which are measured. The processes of introducing the ions into the measuring cell, ion excitation and ion detection are carried out in successive steps of the method.
Since the mass-to-charge ratio of the ions (referred to below simply as “specific mass”, and sometimes simply as “mass”) before the measurement is unknown, the ions are excited by a complete and homogeneous mixture of excitation frequencies. This mixture can be a temporal mixture with frequencies increasing with time (called a “chirp”), or it can be a synchronous, computer-calculated mixture of all frequencies (a “sync pulse”). By specially selecting the phases, the synchronous mixture of the frequencies can be formed so that the amplitudes of the mixture remain restricted to the dynamic region of the digital-to-analog converter, which produces the temporal analog voltage sequence characteristics for the mixture.
The image currents induced by the ions in the detection electrodes are amplified, digitized and analyzed by Fourier analysis for the orbital frequencies present therein. The Fourier analysis transforms the original measurements in the “time domain” into a “frequency domain”, hence the term Fourier transform mass spectrometry (FTMS). The specific masses of the ions and their intensities are then determined from the signals, which can be recognized as peaks in the frequency domain. Owing to the extraordinarily high constancy of the magnetic fields used, and the high accuracy for frequency measurements, it is possible to achieve an extraordinarily accurate mass determination. At present, Fourier transform mass spectrometry is the most accurate of all types of mass spectrometry. Ultimately, the accuracy depends only on the number of ion orbits which can be detected by the measurement.
The longitudinal electrodes usually form a measuring cell with a square or circular cross-section. The cylindrical measuring cell contains four cylinder segments as longitudinal electrodes. Cylindrical measuring cells are the ones most commonly used because they produce the best utilization of the magnetic field, although the image currents of focused clouds of ions with the same mass (image voltages) come close to a rectangular curve.
Since the ions can move freely in the direction of the magnetic field lines and possess, from the filling phase, all velocity components in the direction of the magnetic field, they must be prevented from leaving the measuring cell. To prevent ion losses, the measuring cells are therefore equipped at both ends with electrodes, known as “trapping electrodes”. These are supplied with ion-repelling DC voltage potentials in order to keep the ions in the measuring cell. There are widely differing forms for this electrode pair, the simplest being planar electrodes with a central aperture. The aperture serves to introduce the ions into the measuring cell.
The vacuum in the measuring cell must be as good as possible because, during the measurement of the image currents, the ions should not collide with molecules of residual gas. Each collision of an ion with a molecule of residual gas brings the ion a bit out of the orbiting phase of the other ions with the same specific mass. The loss of phase homogeneity leads to a reduction in the image currents and to a continuous decrease in the signal-to-noise-ratio, which reduces the usable measuring period. The measurement period should amount to at least a few hundred milliseconds, ideally a few seconds. This requires a vacuum in the region of 10−7 to 10−9 Pascal.
Apart from the vacuum, the space charge in the ion cloud can also adversely affect the measurement. The Coulombic repulsion between the ions themselves and, above all, the elastic reflection of the ions moving in the cloud lead to a plurality of disturbances, which also lead to an expansion of the cloud. In present-day instruments, the space charge, alongside the effects of pressure, represents the greatest limitation on achieving a high mass accuracy.
For higher specific ion masses, the decrease in the cyclotron orbital frequency of the ions is inversely proportional to the mass. The resolution, however, is proportional to the number of measured orbits; it is therefore lower for ions of high specific masses than for those of low specific masses, although a high resolution and, correspondingly, a high mass accuracy is particularly desirably for ions of high masses. Since the introduction of ion cyclotron mass spectrometers, repeated attempts have been made to increase the resolution, particularly for higher specific ion masses, by using a larger number of detection electrodes to increase the frequency of the image currents in relation to the cyclotron frequency. If a total of sixteen detection electrodes are used instead of two, then the image currents are each measured sixteen times instead of two times, and the measured frequency increases by a factor of eight. It is to be expected that resolution and mass accuracy are also increased by a factor eight if measured over the same measuring time.
Unfortunately, these experiments have had only moderate success, and so they have regularly been abandoned. The reasons for the moderate success have not been adequately explained. It can be assumed that the ion clouds do not hold together well enough and that, for this reason, they cannot be brought close enough to the detection electrodes. Narrow electrodes require that the ion clouds are brought up very close to the detection electrodes, since otherwise it is scarcely possible to induce the image currents at full strength.
The ion-repelling potentials from the trapping plates form a potential well in the interior of the measuring cell, with a parabolic potential profile along the axis of the measuring cell. The potential profile is only weakly dependent on the shape of these electrodes. The potential profile along the axis is at its minimum at precisely the mid-point of the measuring cell if the ion-repelling potentials across both electrodes have the same value. The ions introduced will therefore execute oscillations in this potential well in the axial direction—so-called trapping oscillations—because they still possess kinetic energy in the axial direction from their introduction. The amplitude of the trapping oscillations depends on their kinetic energy.
The electric field outside the axis of the measuring cell is more complicated to describe. It inevitably contains field components in the radial direction which generate a second type of ion motion: magnetron circular motion. The magnetron gyration is also a circular motion about the axis of the measuring cell, but much slower than the cyclotron circular motion. The additional magnetron circular motion causes the mid-points of the cyclotron circular movements to gyrate around the axis of the measuring cell at the frequency of the magnetron motion, with the result that the trajectory of the ions describes a cycloidal motion.
The superposition of magnetron and cyclotron circular motion is an undesirable phenomenon which leads to a frequency shift in the cyclotron frequency. Furthermore, it leads to a reduction in the usable volume of the measuring cell. The measured frequency of ion motion (the “reduced cyclotron frequency”) is shifted to lower frequencies relative to the unperturbed cyclotron frequency by an amount depending on the cell geometry, the potential on the trapping plates, the magnetic field strength, and the mass of the ion. A measuring cell without magnetron circular motion would be very advantageous because the unperturbed cyclotron frequency could be directly measured and no corrections would have to be applied.
Recently, measuring cells for ion cyclotron resonance mass spectrometry have been elucidated in which practically no magnetron circular motion can develop. (E. Nikolaev, Lecture at the International Mass Spectrometry Conference (IMSC) in Edinburgh, September 2003). In this case, the trapping electrodes are replaced with fine electrode structures, to which an RF voltage is applied and which thus reflect ions of both polarities because of their pseudopotential if the ions possess a specific mass above a mass threshold. The mass threshold can be adjusted by the RF voltage. Electrode structures of this type have been elucidated in U.S. Pat. No. 5,572,035 (J. Franzen). The pseudopotential has a very short range of the order of magnitude of the widths of the structural elements of this electrode structure. The reflection resembles a hard reflection on a matt screen, the scattering effect of the matt screen decreasing as the angle of incidence flattens out.
In U.S. patent application Ser. No. 11/243,510 J. Franzen et al. further the above concept of Nikolaev by applying only DC potentials to the above mentioned fine electrodes. In one embodiment, the fine electrodes take the form of a set of spokes radiating from the axis of the cell. Positive and negative DC potentials are applied to the spokes. The positive and negative potentials are of the same magnitude. The polarity of the DC potential applied to any given spoke is the opposite of that applied to adjacent spokes. The potentials applied to the spokes together with the cyclotron motion of ions in the cell may in some cases result in a pseudopotential that traps the ions in the cell. However, the methods of Nikolaev and Franzen have yet to be experimentally demonstrated and theoretically should work over only a limited mass range. Furthermore, such methods theoretically become less effective as the magnetic field strength is increased.
In other prior art FTICR instruments the above mentioned trapping plates are replaced by cylindrically shaped electrodes of the same inner diameter as the excite/detect electrodes. Ions are trapped by applying a repelling DC potential to the cylinder electrodes. In further prior art “auxiliary” electrodes are used to partially compensate for the effects of the electric field on ion cyclotron motion (A. M Brustkern et al., “A New Electrically Compensated Cylindrical ICR Trap: Procedure for Tuning and Improvements in Mass Resolving Power and Sensitivity”, Proceedings of the 55nd ASMS Conference on Mass Spectrometry and Allied Topics, Indianapolis, Jun. 3-7, 2007. And Y. Naito, Improvement of the Electric Field in the Cylindrical Trapped Ion Cell, Int. J. Mass Spectrom. Ion Processes 120, p179 (1992).). These prior art methods have had limited success.
As discussed below, the reflecting ICR cell according to the present invention overcomes many of the limitations of prior art ICR cells discussed above. The ICR cell disclosed herein provides for the substantial elimination of the radial component of the trapping electric field and thus the magnetron motion of trapped ions.